My final blog post, is a bonus post based on Episode 7 from the podcast Allergies with Ayah, for the scientists out there who are interested in my Masters research project looking at the genetic determinants of food allergy. I completed this in 2020 and due to Covid 19 and the lockdown my project had to be cut short. However, this post gives you a glimpse of the research that I conducted in my Msci year, I describe the aims of the project, with methods and results.
IgE-mediated food allergy is heritable meaning that it has a genetic component to it and is a trait that can be past down from parent to child through genes. It is a complex disease because many factors can contribute to the development of food allergy such as various genes and genetic variants, environmental factors and life style factors. Understanding the genetics can allow us to put prevention strategies in place to reduce those at risk from developing food allergy.
Therefore, my first aim was to perform a literature review to compile a database of potential susceptibility genes and genomic variations. I had to review all the literature that had previously been conducted on this topic and to create a list of potential genetic variations called SNPs. SNP stands for Single Nucleotide Polymorphisms, and these are genomic variants at a single base position in the DNA. SNPs are the most common type of genetic variation among people so are very useful to study when investigating complex diseases.
I essentially trolled the internet and journals searching for papers investigating genes and SNPs associated to IgE-mediated food allergy. I used Medline, Embase and PubMed to in my search strategy.
Search 1 was updated from Suaini et als strategy to collect new publications.
Search 2 I removed age restrictions and included a wider range of allergens.
7 studies were identified using search strategy 1 and 15 studies using search strategy 2.
20 studies were included from Sauini et al review. I identified 241 SNPs in 46 gene regions that were associated with IgE-food allergy. I then created a database of everything I found. I found that many papers investigated SNP’s within the human leukocyte antigen or HLA locus. The HLA locus is a complex of genes on chromosome 6 in humans which codes for a group of proteins found on the surfaces of cells that help the immune system recognize foreign substances. My search also identified variations in the FLG gene which codes for filaggrin, a structural protein involved in maintaining the skins structural integrity. As well other gene regions which code for proteins involved in the immune response. I talk about some of these genes and regions in episode 3 which dives into the risk factors of developing food allergy so if you are interested in learning more about them then head over to that episode or my blog post about it. Overall, the HLA gene was most commonly investigated followed by FLG, STAT6 IL4, IL10, CD14 and C11orf30.
For the candidate gene study, I investigated this database of SNP’s and genes in a cohort who are food or inhalant sensitised or atopic. This cohort were fully genotyped and came from the Manchester Asthma and Allergy Study (MAAS). I investigated the list of SNPs and genes by using various bioinformatic tools.
Prior to this study, additional genotypes for the MAAS cohort were imputed. I used SNPTEST for frequentist association testing and ranked outputs based on p value, info scores and allele frequency.
The functional properties of the top SNPs were annotated in the VEP.
Locuszoom and LDproxy were used to visualise SNPs in LD with candidate SNPs.
Missense variants were analysed using Polyphen-2 and SIFT.
I identified serval variations associated to allergy and inhalant sensitisation in the cohort.
I identified mostly intronic SNPs associated to sensitisation in the MAAS cohort.
There were more cases compared to controls who had the candidate SNPs for any atopic and food sensitised age 5 suggesting these SNPs were contributing to sensitisation in a deleterious manner. Whilst there were more controls with the inhalant sensitised age 8 SNP suggesting it had protective effect on the phenotype. Most SNPs in high linkage disequilibrium (LD) with the candidate SNPs were intron variants. Intron variants could have affected alternative splicing of the mRNA. However, without further investigation by qpCR and western blot it was difficult to know their effect on protein function.
We identified a damaging with confidence missense SNP in high LD with the top SNP for inhalant sensitisation at age 5 which we presumed was more likely contributing to the phenotype.
The most interesting SNP I identified was a damaging missense variant in high LD with a SNP in the Interleukin 1 Receptor Like (IL1RL1) gene. This is a receptor situated on the surface of effector cells, involved in inflammatory signalling. IL1RL1 is a highly researched gene associated with asthma and variations in the gene were found to be associated to inhalant sensitization in our cohort. This missense SNP causes a glutamine to leucine amino acid substitution. Glutamine is a polar amino acid and frequently appears in the binding site of receptors. Leucine is non-polar and unreactive thus its biologically plausible that this substitution could alter the receptors, ligand recognition ability.
Overall given more time, a TaqMan genotyping assays would have been developed for replication in an independent food allergic cohort from the EuroPrevall study. Future, studies should combine our genomic data with transcriptome data to understand how these SNPs impact IgE-food allergy aetiology.
Acknowledgement
I would like to thank; Professor Clare Mills, Dr John Curtin and Dr Elaine Gauson and the Mills Lab group without whom this project would not be possible.
References:
Custovic A, Simpson BM, Murray CS, Lowe L, Woodcock A, NAC Manchester Asthma and Allergy Study Group. The national asthma campaign Manchester asthma and allergy study. Pediatric Allergy and Immunology. 2002 Dec;13:32-7.
Betts, M.J. and Russell, R.B., (2003). Amino acid properties and consequences of substitutions. Bioinformatics for geneticists, 317, p.289
Moffatt, M. F., Gut, I. G., Demenais, F., Strachan, D. P., Bouzigon, E., Heath, S., Von Mutius, E., Farrall, M., Lathrop, M. & Cookson, W. O. C. M. (2010). 'A large-scale, consortium-based genomewide association study of asthma', New England Journal of Medicine, 363(13), pp. 1211-1221.
Xepapadaki P, Fiocchi A, Grabenhenrich L, Roberts G, Grimshaw KE, Fiandor A, Larco JI, Sigurdardottir S, Clausen M, Papadopoulos NG, Dahdah L. Incidence and natural history of hen's egg allergy in the first 2 years of life—the EuroPrevall birth cohort study. Allergy. 2016 Mar;71(3):350-7.
Suaini, N. H. A., Wang, Y., Soriano, V. X., Martino, D. J., Allen, K. J., Ellis, J. A. & Koplin, J. J. (2019). 'Genetic determinants of paediatric food allergy: A systematic review', Allergy, 74(9), pp. 1631-1648.
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